Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing.
ABSTRACT: Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ?2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [(15)N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using large-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in (15)N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.
Project description:Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of petroleum pollution, however relatively little is known about how these interactions are influenced by petroleum pollution. In this experimental study using a microcosm approach, we examined how plant ecophysiological traits, soil nutrients and microbial activities were influenced by petroleum pollution in Phragmites australis, a phytoremediating species. Generally, petroleum pollution reduced plant performance, especially at early stages of plant growth. Petroleum had negative effects on the net accumulation of inorganic nitrogen from its organic forms (net nitrogen mineralization (NNM)) most likely by decreasing the inorganic nitrogen available to the plants in petroleum-polluted soils. However, abundant dissolved organic nitrogen (DON) was found in petroleum-polluted soil. In order to overcome initial deficiency of inorganic nitrogen, plants by dint of high colonization of arbuscular mycorrhizal fungi might absorb some DON for their growth in petroleum-polluted soils. In addition, through using a real-time polymerase chain reaction method, we quantified hydrocarbon-degrading bacterial traits based on their catabolic genes (i.e. alkB (alkane monooxygenase), nah (naphthalene dioxygenase) and tol (xylene monooxygenase) genes). This enumeration of target genes suggests that different hydrocarbon-degrading bacteria experienced different dynamic changes during phytoremediation and a greater abundance of alkB was detected during vegetative growth stages. Because phytoremediation of different components of petroleum is performed by different hydrocarbon-degrading bacteria, plants' ability of phytoremediating different components might therefore vary during the plant life cycle. Phytoremediation might be most effective during the vegetative growth stages as greater abundances of hydrocarbon-degrading bacteria containing alkB and tol genes were observed at these stages. The information provided by this study enhances our understanding of the effects of petroleum pollution on plant-microbe interactions and the roles of these interactions in the phytoremediation of petroleum-polluted soil.
Project description:The aim of work was to check and make comparison of efficacy for five approaches for petroleum crude contaminated agricultural soil remediation by making use of soil microcosms. Concerning the published literature in our information, this is the first report comparing five approaches i.e. abiotic losses, native microbial flora, nutrient amendments and pre-adapted native microbial culture and concurrent amendments of nutrients?+?pre-adapted native microbial culture for agricultural soil bioremediation using <i>Pseudomonas aeruginosa</i> NCIM 5514 by performing soil microcosm experiments. 96.00?±?0.18% degradation of petroleum hydrocarbon fractions in 60 days of the experiment was observed when nutrients and <i>P. aeruginosa</i> NCIM 5514 were applied concomitantly. In nutrients- and <i>P. aeruginosa</i> NCIM 5514-added microcosm reduction in nitrogen, organic carbon, and phosphorus was noted. <i>P. aeruginosa</i> NCIM 5514, can be applied as a prospective bioremediation agent to remediate petroleum crude contaminated soil.
Project description:Due to the increasing pollution by petroleum hydrocarbons (PHs), it is an important task to develop eco-friendly and highly efficient methods for remediating petroleum-contaminated soils. In this study, bioremediation technology was applied to remediate PHs contaminated soils, and the bacterial community structure and physicochemical characteristics of the soil treated using different bioremediation regimens were analyzed. Compared with the control condition (S0), the PHs removal efficiency of biostimulation (S2) and bioaugmentation (S3) was increased significantly. Combined biostimulation with bioaugmentation (S4) had the highest PHs removal efficiency, up to 60.14 ± 4.12%. Among all the selected remediation strategies (S1-S4, S1: soil moisture content: 25-30%), the bacterial alpha-diversity was higher than in S0. The genera Acinetobacter, Escherichia-Shigella, Bacteroides, Microbacterium, and Parabacteroides were found to greatly contribute to PHs' degradation. In the group S4, the PH-degraders and soil enzyme activity were higher than in the other remediation regimens, and these indices gradually decreased in the mid-to-later periods of all remediation tests. Additionally, the abundance of alkB and nah genes was increased by improving the environmental condition of the microorganism communities. Redundancy analysis (RDA) revealed that the total nitrogen (TN) and total phosphorus (TP) had a positive correlation with total PHs degradation. This study offers insights into the microbial community response to environmental factors during bioremediation, which shows a promoting effect in enhancing the efficiency of PHs remediation.
Project description:Increased exploration and exploitation of resources in the Arctic is leading to a higher risk of petroleum contamination. A number of Arctic microorganisms can use petroleum for growth-supporting carbon and energy, but traditional approaches for stimulating these microorganisms (for example, nutrient addition) have varied in effectiveness between sites. Consistent environmental controls on microbial community response to disturbance from petroleum contaminants and nutrient amendments across Arctic soils have not been identified, nor is it known whether specific taxa are universally associated with efficient bioremediation. In this study, we contaminated 18 Arctic soils with diesel and treated subsamples of each with monoammonium phosphate (MAP), which has successfully stimulated degradation in some contaminated Arctic soils. Bacterial community composition of uncontaminated, diesel-contaminated and diesel+MAP soils was assessed through multiplexed 16S (ribosomal RNA) rRNA gene sequencing on an Ion Torrent Personal Genome Machine, while hydrocarbon degradation was measured by gas chromatography analysis. Diversity of 16S rRNA gene sequences was reduced by diesel, and more so by the combination of diesel and MAP. Actinobacteria dominated uncontaminated soils with <10% organic matter, while Proteobacteria dominated higher-organic matter soils, and this pattern was exaggerated following disturbance. Degradation with and without MAP was predictable by initial bacterial diversity and the abundance of specific assemblages of Betaproteobacteria, respectively. High Betaproteobacteria abundance was positively correlated with high diesel degradation in MAP-treated soils, suggesting this may be an important group to stimulate. The predictability with which bacterial communities respond to these disturbances suggests that costly and time-consuming contaminated site assessments may not be necessary in the future.
Project description:A microcosm experiment was conducted for 112 d by spiking petroleum hydrocarbons into soils from four regions of China. Molecular analyses of soils from microcosms revealed changes in taxonomic diversity and oil catabolic genes of microbial communities. Degradation of total petroleum hydrocarbons (TPHs) in Sand from the Bohai Sea (SS) and Northeast China (NE) exhibited greater microbial mineralization than those of the Dagang Oilfield (DG) and Xiamen (XM). High-throughput sequencing and denaturing gradient gel electrophoresis (DGGE) profiles demonstrated an obvious reconstruction of the bacterial community in all soils. The dominant phylum of the XM with clay soil texture was Firmicutes instead of Proteobacteria in others (DG, SS, and NE) with silty or sandy soil texture. Abundances of alkane monooxygenase gene AlkB increased by 10- to 1000-fold, relative to initial values, and were positively correlated with rates of degradation of TPHs and n-alkanes C13-C30. Abundances of naphthalene dioxygenase gene Nah were positively correlated with degradation of naphthalene and total tricyclic PAHs. Redundancy analysis (RDA) showed that abiotic process derived from geographical heterogeneity was the primary effect on bioremediation of soils contaminated with oil. The optimization of abiotic and biotic factors should be the focus of future bioremediation of oil contaminated soil.
Project description:The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with the unplanted system. Among the representative alkane hydroxylase genes alkB, CYP153, almA and ladA, the first two were detected in this study, and the genotypes of both genes were apparently different among the systems studied. Their diversity was also higher on the rhizoplanes of the grasses than in unplanted oil-contaminated soils. Actinobacteria-related genes in particular were among the most diverse alkane hydroxylase genes on the rhizoplane in this study, indicating that they are one of the main contributors to degrading alkanes in oil-contaminated soils during phytoremediation. Actinobacteria-related alkB genes and CYP153 genes close to the genera Parvibaculum and Aeromicrobium were found in significant numbers on the rhizoplanes of grasses. These results suggest that the increase in diversity and genotype differences of the alkB and CYP153 genes are important factors affecting petroleum hydrocarbon-degrading ability during phytoremediation.
Project description:Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).
Project description:Most components of petroleum oily sludge (POS) are toxic, mutagenic and cancer-causing. Often bioremediation using microorganisms is hindered by the toxicity of POS. Under this circumstance, phytoremediation is the main option as it can overcome the toxicity of POS. Cajanus cajan a legume plant, was evaluated as a phyto-remediating agent for petroleum oily sludge-spiked soil. Culture dependent and independent methods were used to determine the rhizosphere microorganisms' composition. Degradation rates were estimated gravimetrically. The population of total heterotrophic bacteria (THRB) was significantly higher in the uncontaminated soil compared to the contaminated rhizosphere soil with C. cajan, but the population of hydrocarbon-utilizing bacteria (HUB) was higher in the contaminated rhizosphere soil. The results show that for 1 to 3% oily sludge concentrations, an increase in microbial counts for all treatments from day 0 to 90 d was observed with the contaminated rhizosphere CR showing the highest significant increase (p ?<?0.05) in microbial counts compared to other treatments. The metagenomic study focused on the POS of 3% (w/w) and based on the calculated bacterial community abundance indices showed an increase in the values for Ace, Cho, Shannon (Shannon-Weaver) and the Simpson's (measured as InvSimpson) indices in CR3 compared to CN3. Both the Simpson's and the Shannon values for CR3 were higher than CN3 indicating an increase in diversity upon the introduction of C. cajan into the contaminated soil. The PCoA plot revealed community-level differences between the contaminated non-rhizosphere control and contaminated rhizosphere microbiota. The PCoA differentiated the two treatments based on the presence or absence of plant. The composition and taxonomic analysis of microbiota-amplified sequences were categorized into eight phyla for the contaminated non-rhizosphere and ten phyla for the contaminated rhizosphere. The overall bacterial composition of the two treatments varied, as the distribution shows a similar variation between the two treatments in the phylum distribution. The percentage removal of total petroleum hydrocarbon (TPH) after 90 days of treatments with 1, 2, 3, 4, and 5% (w/w) of POS were 92, 90, 89, 68.3 and 47.3%, respectively, indicating removal inhibition at higher POS concentrations. As the search for more eco-friendly and sustainable remediating green plant continues, C. cajan shows great potential in reclaiming POS contaminated soil. Our findings will provide solutions to POS polluted soils and subsequent re-vegetation.
Project description:Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring ?-proteobacteria like Shewanella or Hydrocarboniphaga as well as ?-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and might be a useful agent to stimulate bioremediation of hydrocarbons in contaminated soils.
Project description:Pseudomonas microorganisms are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils were shown to affect residual hydrocarbons in the course of biodegradation. The regulatory mechanisms of expression of hydrocarbon catabolic genes in low nitrogen/low iron conditions remain unclear. The catabolism of naphthalene, a two-ring polycyclic aromatic hydrocarbon, has been well studied in pseudomonads in terms of the involvement of specific transcriptional activators, thus making it useful in revealing additional regulatory control of the adaptation of hydrocarbon destructors to a low level of the essential nutrients. The Pseudomonas putida strain BS3701 is a component of the "MicroBak" preparation for soil remediation. Previously, this strain was shown to contain genes encoding the key enzymes for naphthalene catabolism: naphthalene 1,2-dioxygenase, salicylate hydroxylase, catechol 2,3-dioxygenase, and catechol 1,2-dioxygenase. Our study aimed to clarify whether the naphthalene catabolic gene expression is dependent on the amount of nitrogen and iron in the growth culture medium, and if so, at exactly which stages the expression is regulated. We cultivated the strain in low nitrogen/low iron conditions with the concurrent evaluation of the activity of the key enzymes and the mRNA level of genes encoding these enzymes. We are the first to report that naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation.